Pantothenic acid is a vitamin of commercial importance which is used in cosmetics, medicine, human nutrition and animal nutrition.
Pantothenic acid can be prepared by chemical synthesis, or biotechnologically by the fermentation of suitable microorganisms in suitable nutrient solutions. In the chemical synthesis, DL-pantolactone is an important compound. It is prepared in a multi-stage process from formaldehyde, isobutyraldehyde and cyanide. In further process steps, the racemic mixture is separated, D-pantolactone is subjected to a condensation reaction with xcex2-alanine, and D-pantothenic acid is obtained.
An advantage of the fermentative preparation by microorganisms is the direct formation of the desired stereoisomeric D-form.
Various types of bacteria, such as, for example, Escherichia coli, Arthrobacter ureafaciens, Corynebacterium erythrogenes, Brevibacterium ammoniagenes, and also yeasts, such as, for example, Debaromyces castellii, can produce D-pantothenic acid in a nutrient solution which comprises glucose, DL-pantoic acid and xcex2-alanine, as shown in EPA 0 493 060. EPA 0 493 060 furthermore shows that in the case of Escherichia coli, the formation of D-pantothenic acid is improved by amplification of pantothenic acid biosynthesis genes contained on the plasmids pFV3 and pFV5, in a nutrient solution comprising glucose, DL-pantoic acid and xcex2-alanine. EPA 0 590 857 and U.S. Pat. No. 5,518,906 describe mutants derived from the Escherichia coli strain IFO3547, such as FV5714, FV525, FV814, FV521, FV221, FV6051 and FV5069, which carry resistances to various antimetabolites, such as salicylic acid, xcex2-ketobutyric acid, xcex2-hydroxyaspartic acid, O-methylthreonine and xcex1-ketoisovaleric acid and produce pantoic acid in a nutrient solution comprising glucose, and D-pantothenic acid in a nutrient solution comprising glucose and xcex2-alanine. It is furthermore shown in EPA 0 590 857 and U.S. Pat. No. 5,518,906 that after amplification of the pantothenic acid biosynthesis genes contained on the plasmid pFV31 in the abovementioned strains, the production of D-pantoic acid in a nutrient solution comprising glucose and the production of D-pantothenic acid in a nutrient solution comprising glucose and xcex2-alanine is improved.
In addition, WO 97/10340 shows that in strains of Escherichia coli which form pantothenic acid, pantothenic acid production can be increased further by increasing the activity of the enzyme acetohydroxy acid synthase II, an enzyme of valine biosynthesis.
It is an object of the present invention to provide an improved process for the preparation of pantothenic acid.
The vitamin pantothenic acid is a product of commercial importance which is used in cosmetics, medicine, human nutrition and animal nutrition. There is therefore a general interest in providing improved processes for the preparation of pantothenic acid. When D-pantothenic acid or pantothenic acid or pantothenate are mentioned in the present application, they are intended to include not only the free acid but also the salts of D-pantothenic acid, such as, for example, the calcium, sodium, ammonium or potassium salt.
The invention provides a process for the preparation and improvement of pantothenic acid-producing microorganisms by amplification, in particular over-expression, of nucleotide sequences which code for ketopantoate reductase, in particular sequences of the panE gene, individually or in combination with one another, and optionally, in addition, sequences of the ilvC gene.
The term xe2x80x9camplificationxe2x80x9d in this connection is intended to mean an increase in the intracellular activity of one or more enzymes which are coded by the corresponding DNA by increasing the number of copies of the gene(s), using a potent promoter or a gene which codes for a corresponding enzyme having a high specific activity, and optionally combining these measures.
In particular, it has been found that over-expression of the panE gene together with the genes panB, panC and panD, further improves the formation of pantothenic acid. To achieve the over-expression, the number of copies of the corresponding genes can be increased by means of plasmid vectors, such as, for example, pBR322 (Subcliffe, COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY 1979, 43: 77-90) or pUC19 (Viera, Gene 1982 19:259-268), or the promoter and regulation region upstream of the structural gene can be mutated. A known example of this is the lac-UV5 mutation of the lac promoter (Winnacker: Gene and Klone, Eine Einfxc3xchrung in die Gentechnologie [From Genes to Clones, Introduction to Gene Technology (Verlag Chemie, Weinheim, Germany, 1990). Expression cassettes which are incorporated upstream of the structural gene act in the same way. This method has been used, for example, by LaVallie et al. (BIO/TECHNOLOGY 11, 187-193 (1993) and in PCT/US97/13359. Alternatively, over-expression of the genes in question can be achieved by changing the composition of the media and the culture procedure. An example of this is the universally known regulation of the expression of the lac operon by glucose and lactose. The present inventors moreover have found that over-expression of the panE gene has an advantageous effect in strains which have resistance mutations to metabolites and antimetabolites, such as, for example, resistance to L-valine. If has furthermore been found that over-expression of the panE gene has an advantageous effect in strains which have defect mutations in genes of metabolic routes, such as, for example, the avtA or ilvE gene, which convert precursors of pantothenic acid or reduce the formation of pantothenic acid.
The microorganisms to which the present invention relates can synthesize pantothenic acid from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. These are fungi, yeasts or, in particular. Gram-positive bacteria, for example, of the genus Corynebacterium, or Gram-negative bacteria, such as, for example, those of the Enterobacteriaceae. Of the family of the Enterobacteriaceae, the genus Escherichia with the species Escherichia coli may be mentioned in particular. Within the species Escherichia coli there may be mentioned the so-called K-12 strains, such as, for example, the strains MG1655 or W3110 (Neidhard et al.: Escherichia coli and Salmonella. Cellular and Molecular Biology (ASM Press, Washington D.C.)) or the Escherichia coli wild type strain IFO3547 (Institute of Fermentation, Osaka, Japan) and mutants derived from these. Of the genus Corynebacterium, the species Corynebacterium glutamicum, which is known among specialists for its ability to form amino acids, is of particular interest. This species includes wild type strains, such as, for example, Corynebacterium glutamicum ATCC13032, Brevibacterium flavum ATCC14067, Corynebacterium melassecola ATCC17965 and others.
To isolate the ilvC gene and the panE gene, a mutant of, for example, Escherichia coli which carries a mutation in the ilvC gene and panE gene, is first prepared.
The nucleotide sequence of the ilvC gene of Escherichia coli is known (Wek and Hatfield, Journal of Biological Chemistry 261, 2441-2450 (1986)). Methods for isolation of chromosomal DNA are also known (Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). By choosing suitable primers, the ilvC gene can be amplified with the aid of the polymerase chain reaction (Innis et al., PCR protocols. A guide to methods and applications, 1990, Academic Press). It is then introduced into a plasmid vector. Possible plasmid vectors are those which can replicate in the corresponding microorganisms. For Escherichia coli, for example, the vectors pSC101 (Vocke and Bastia, Proceedings of the National Academy of Science U.S.A. 80 (21), 6557-6561 (1983)) or pKK223-3 (Brosius and Holy, Proceedings of the National Academy of Science USA 81, 6929 (1984)), for Corynebacterium glutamicum, for example, the vector pJC1 (Cremer et al., Mol. Gen. Genet. 220:478-480 (1990)) or pEKEx2 (Eikmanns et al., Gene 102:93-98 (1991)) or pZ8-1 (European Patent Specification 0 375 889) and for Saccharomyces cerevisiae, for example, the vector pBB116 (Berse, Gene 25: 109-117 (1983)) or pDG1 (Buxton et al., Gene 37: 207-214 (1985)) are possible for the present invention. Methods for incorporation of DNA fragments into plasmid vectors are described by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). Methods for transformation and electroporation are described by Tauch et al. (FEMS Microbiology Letters 123:343-347 (1994)). A example of such a transformed strain is the Escherichia coli strain MG1655/pFE32. Plasmid pFE32 contains the ilvC gene of MG1655 which has been incorporated into the vector pBR322. Another example of such a transformed strain is the Corynebacterium glutamicum strain ATCC13032/pFE91. Plasmid pFE91 contains the ilvC gene of ATCC13032 which has been incorporated into the vector pECm3. Plasmid pECm3 is a derivative of plasmid pECm2 (Tauch, 1994, FEMS Microbiological Letters, 123:343-348), the kanamycin resistance gene of which has been removed by a BglII and BamHI restriction with subsequent re-ligation.
For incorporation of a mutation into the ilvC gene which eliminates its function, for example, a deletion or insertion can be used. To generate a deletion, an internal part of the nucleotide sequence of the structural gene can be removed with the aid of suitable restriction enzymes and subsequent linking of the ends formed. The ilvC gene mutated in this manner has no function. A second gene which codes for a resistance to an antibiotic can be incorporated into the ilvC gene in the same manner. The ilvC gene mutated in this manner also has no function. The ilvC gene mutated in this manner can then be introduced into a microorganism to replace the wild type gene in the chromosome thereof. Methods of how to carry out this gene exchange are known in the literature. For Escherichia coli, the method described by Hamilton et al. (Journal of Bacteriology 171, 4617-4622 (1989)), which is based on temperature-sensitive replication mutants of the plasmid pSC101, can be employed. pMAK705 is an example of such a plasmid. For Corynebacterium glutamicum, the method of gene exchange described by Schwarzer and Pxc3xchler (BIO/TECHNOLOGY 9, 84-87 (1991)), in which non-replicative plasmid vectors are used, can be used. For Saccharomyces cerevisiae a method of controlled gene exchange is described by Roca et al. (Nucleic Acid Research 20(17), 4671-4672 (1992)).
A mutated ilvC gene can be prepared, for example, from a wild type ilvCxe2x88x92 gene as follows. Plasmid pFE32 comprised of pBR322, is incorporated into the BamHI restriction cleavage site of the ilvC wild type gene. The aacC1 gene, which codes for resistance to the antibiotic gentamycin, was incorporated into the KpnI cleavage site of the ilvC gene of pFE32 (Schweizer, Bio Techniques 15 (5), 831-834 (1993)). The plasmid pFE33 obtained in this manner contains the ilvC::aacC1 allele, which can no longer form functional ilvC gene product. The ilvC::aacC1 allele was removed from the plasmid pFE33 and introduced into the SphI cleavage site of the plasmid pMAK705, as a result of which the plasmid pDB1 was formed. Plasmid pDB1 is a plasmid vector which is capable of allele exchange and comprises on the one hand pMAK705 and on the other hand the ilvC::aacC1 allele. Plasmid pDB1 was used in the method described by Hamilton et al. to exchange the wild type ilvC gene present in MG 1655 for the ilvC::aacC1 allele. The strain formed in this manner is designated FE4.
To isolate a mutant of FE4 which carries a mutation in the panE gene, the strain FE4 was subjected to a transposon mutagenesis with the transposon Tn5. Transposon Tn5 is described by Auerswald (COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY 45, 107-113 (1981)). The method of transposon mutagenesis is described, for example, in the handbook by Miller, A: Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria (Cold Spring Harbor Laboratory Press, 1992). The method is furthermore described by Simon (Gene 80, 161-169 (1998)). and also in the handbook by Hagemann: Gentechnologische Arbeitsmethoden [Working Methods of Genetic Engineering] (Gustav Fischer Verlag, 1990) and in numerous other publications accessible to the public. Mutants can also be produced after mutagenesis with ultraviolet light or after treatment with a mutation-inducing chemical, such as, for example, N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine. Among the mutants obtained in this manner, after testing the growth substance requirements, in particular the pantothenic acid requirement, those mutants which carry a mutation in a gene of pantothenic acid biosynthesis can be isolated. Those mutants in need of pantothenic acid which can utilize not ketopantoate but pantoate as a growth substance and are therefore mutated in the panE gene which codes for ketopantoate reductase (EC 1.1.1169) are of particular interest. An example of this is the strain FE5 obtained in this manner, which, in addition to the ilvC::aacC1 mutation, carries a panE::Tn5 mutation.
Microorganisms which carry a defect mutation in the ilvC and panE gene, such as, for example, Escherichia coli strain FE5, can be used as cloning hosts for isolation of the ilvC gene and of the particularly interesting panE gene, or of nucleotide sequences which code for proteins with ketopantoate reductase activity.
A gene library of the microorganisms of interest was created in this context. The construction of gene libraries is described in generally known textbooks and handbooks. The textbook by Winnacker: Gene and Klone, Eine Einfxc3xchrung in die Gentechnologie [From Genes to Clones, Introduction to Gene Technology] (Verlag Chemie, Weinheim, Germany, 1990) or the handbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may be mentioned, for example. A known gene library is that of the E. coli K-12 strain W3110 described by Kohara et al. (Cell 50, 495-508 (1987)). It has since become possible to acquire gene libraries of various microorganisms commercially, such as, for example, a gene library of Saccharomyces pombe strain Sp63 from Stratagene (Heidelberg, Germany) in the plasmid lambda FIX II (Elgin, Strategies 4: 6-7(1991)), a gene library of the Escherichia coli strain W1485 from CLONTECH (Heidelberg, Germany) in the plasmid pGAD10 (Kitts, CLONTECH (Heidelberg, Germany) Vectors On Disc version 1.3, 1994), the nucleotide sequence of which is accessible under the GenBank accession number U13188. The gene library prepared in the manner described above can then be introduced by transformation into the host FE5 described above. By way of example, the pGAD10 gene library of W1485 was thus introduced into the strain FE5 by transformation, and the resulting transformants were investigated for their ability to grow on a pantothenic acid-free nutrient medium. The insertions contained in the plasmid DNA of the resulting pantothenic acid-prototrophic transformants can be investigated by determination of the nucleotide sequence. Methods for determination of nucleotide sequences can be found, for example, in Sanger et al. (Proceedings of the National Academy of Science USA 74:5463-5467 (1977)). Nucleotide sequences can be assigned to genes by means of homology investigations. One possibility for this homology search is comparison with nucleotide sequences of the EMBL and GenBank databanks, which can be carried out by means of the BLAST E-mail Service (Altschul, Journal of Molecular Biology 215, 403-410 (1990)). An example of such a transformant is the strain FE5/pFEbank16 which carries the panE gene of the E. coli strain MG1655.
The panE gene isolated and identified in the manner described can then be expressed in a desired microorganism. For this purpose, it is amplified by plasmid vectors. These in turn can be equipped with signal structures, which ensure efficient transcription and translation. An overview of expression vectors is to be found, for example, in the textbook by Winnacker: Gene and Klone, Eine Einfxc3xchrung in die Gentechnologie [From Genes to Clones, Introduction to Gene Technology] (Verlag Chemie, Weinheim, Germany, 1990) or in Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). Expression signals, such as, for example, the tac promoter, can furthermore be incorporated into the chromosome upstream of the panE gene. Such methods are described in WO 98/04715. The panE gene to be expressed can be removed from the cloned chromosomal DNA fragment, or it can be amplified in turn with the aid of the polymerase chain reaction. The amount of ketopantoate reductase present in the microorganism in question can be determined with the aid of the method described by Shimizu et al. (Journal of Biological Chemistry 263: 12077-12084 (1988)). A example of such a strain is the Escherichia coli strain MG1655/pFE65. Plasmid pFE65, comprising the vector pKK223-3, has been incorporated into the EcoRI restriction cleavage site of the panE gene of Escherichia coli MG1655.
According to the invention, it has proved advantageous to amplify, in particular to over-express, one or more genes of pantothenic acid biosynthesis in addition to the panE gene, which codes for ketopantoate reductase. These include the genes which code for the enzymes ketopantoate hydroxymethyltransferase (EC 4.1.2.12), aspartate 1-decarboxylase (EC 4.1.1.11) and pantothenate synthetase (EC 6.3.2.1). In Escherichia coli, these genes are designated panB, panD and panC (Miller, A Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria (Cold Spring Harbor Laboratory Press, 1992)). For this, the genes can be incorporated into various compatible plasmid vectors. Examples of these are described by Bartolome et al. (Gene 102, 75-78 (1991). Gene expression can furthermore be increased by changing the chromosomal signal structures lying upstream. The genes in question can, moreover, be placed under the control of a common promoter, in an arrangement in succession, and incorporated into a plasmid vector and introduced into a suitable microorganism. An example of this is Escherichia coli strain MG1655/pFE80. The plasmid pFE80 comprises the plasmid pKK223-3, which contains the genes panB, panD, panC and panE in the stated sequence. The tac promoter is contained in pFE80 as an expression signal upstream of the panB gene.
It has also proved advantageous to over-express the panE gene and the expression unit consisting of the genes panB, panD, panC and panE in host strains which contain chromosomal mutations.
It is possible to use mutations, individually or together, which produce resistances to metabolism products, such as, for example, L-valine or xcex1-ketoisovaleric acid, or to analogues of metabolism products, such as, for example, xcex2-hydroxyaspartic acid or O-methylthreonine. Such mutants occur spontaneously or can be produced by mutagenesis with ultraviolet light or treatment with a mutation-inducing chemical, such as, for example, N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine, and can then be selected or agar plates containing the appropriate substance. Processes for inducing mutation and for selection are generally known and can be found, inter alia, in Miller (A Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria (Cold Spring Harbor Laboratory Press, 1992)) or in the handbook xe2x80x9cManual of Methods for General Bacteriologyxe2x80x9d of the American Society for Bacteriology (Washington D.C., USA). An example of such a mutant is Escherichia coli strain FE6, which has been isolated as spontaneously occurring, L-valine-resistant mutant of the strain MG1655.
Adverse or troublesome chromosomally coded metabolism reactions can furthermore be eliminated in a controlled manner. For this, insertions or deletions are introduced into the corresponding genes and the mutated genes or alleles formed in this manner are incorporated into the chromosome of the host. The methods which have been described above for mutation of the ilvC gene can be employed. An example of such a mutant is the Escherichia coli strain FE7, which carries an avtA::aadB mutation in the chromosome. This is the strain MG1655, in which the aadB gene from plasmid pHP45xcexa9, which imparts resistance to streptomycin, has been introduced into the avtA gene (Prentki and Krisch, Gene 29, 303-313 (1984)). The panE gene can then be over-expressed in the host strains prepared in this manner, either alone or in combination with other genes. Examples of these are the strains FE6/pFE80 and FE7/pFE80.
The microorganisms prepared according to the invention can be cultured continuously or discontinuously in the batch process or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of pantothenic acid production. A summary of known culture methods is described in the textbook by Chmiel (Bioprozesstechnik 1. Einfxc3xchrung in die Bioverfahrenstechnik [Bioprocess Technology 1. Introduction to Bioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and Peripheral Equipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)). The culture medium to be used must meet the requirements of the particular microorganisms. Descriptions of culture media for various microorganisms are contained in the handbook xe2x80x9cManual of Methods for General Bacteriologyxe2x80x9d of the American Society for Bacteriology (Washington D.C., USA, 1981). Sugars and carbohydrates, such as, for example, glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as, for example, soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as e.g. palmitic acid, stearic acid and linoleic acid, alcohols, such as, for example, glycerol and ethanol, and organic acids, such as, for example, acetic acid, can be used as the source of carbon. These substances can be used individually or as a mixture. Organic nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or inorganic compounds, such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen. The sources of nitrogen can be used individually or as a mixture. Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the source of phosphorus. The culture medium must furthermore comprise salts of metals, such as, for example, magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, can be employed in addition to the abovementioned substances. Precursors of pantothenic acid, such as xcex2-alanine or ketopantoic acid and salts thereof, can also be added to the culture medium. The starting substances mentioned can be added to the culture in the form of a single batch, or can be added during the cultivation in a suitable manner.
Basic compounds, such as sodium hydroxide, potassium hydroxide and ammonia, or acid compounds, such as phosphoric acid and sulfuric acid, can be used to control the pH of the culture. Antifoams, such as, for example, fatty acid polyglycol esters or silicone oils, can be employed to control the development of foam. Suitable substances having a selection action, for example, antibiotics, can be added to the medium to maintain the stability of plasmids. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as, for example, air, are introduced into the culture. The temperature of the culture is usually 20xc2x0 C. to 50xc2x0 C., and preferably 25xc2x0 C. to 45xc2x0 C. Culturing is continued until a maximum of pantothenic acid has formed. This target is usually reached within 10 hours to 160 hours.
The concentration of pantothenic acid formed can be determined by known processes (Velisek; Chromatographic Science 60, 515-560 (1992)).
The following microorganisms have been deposited at the Deutsche Sammlung fur Mikrorganismen and Zellkulturen (DSMZ=German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty:
Escherichia coli K12 strain FE5 as DSM12378 on Aug. 18, 1998
Escherichia coli K12 strain MG1655/pFE32 as DSM12413 on Sep. 16, 1998
Escherichia coli K12 strain MG1655/FE65 as DSM12382 on Aug. 18, 1998
Escherichia coli K12 strain MG1655/FE80 as DSM12414 on Sep. 16, 1998
Escherichia coli K12 strain FE6 as DSM12379 on Aug. 18, 1998
Escherichia coli K12 strain FE7 as DSM12380 on Aug. 18, 1998
The process according to the invention provides the person skilled in the art with a new tool for improving the formation of pantothenic acid by microorganisms in a controlled manner.